Treatment for dupuytren&#39;s disease

ABSTRACT

Musculoskeletal fibroproliferative disorders, such as Dupuytren&#39;s disease may be treated by administering locally a TNF-α antagonist. TNF-α antagonists find particular utility in inhibiting the progression of early disease state Dupuytren&#39;s disease and other musculoskeletal fibroproliferative disorders and, in combination with extracellular matrix degradation agents (such ascollagenase or matrix metalloproteinase I), treating advanced disease state Dupuytren&#39;s disease and, in particular inhibiting recurrence.

This is a national stage application of PCT/EP2011/069147, filedinternationally on Oct. 31, 2011, which claims priority to GB 1018325.9,filed Oct. 30, 2010; GB 1018362.2, filed Nov. 1, 2010 and GB 1113718.9,filed Aug. 10, 2011.

FIELD OF THE INVENTION

This invention relates to the treatment of musculoskeletalfibroproliferative disorders such as fibromatosis and, in particular,Dupuytren's disease. In particular it relates to a composition ortherapeutic agent or to a combination of such compositions ortherapeutic agents for the treatment, prophylaxis or prevention ofprogression of musculoskeletal fibroproliferative disorders, especiallyDupuytren's disease, to the use of such composition/therapeutic agent orcombination of compositions/therapeutic agents for the treatment,prophylaxis or prevention of progression of musculoskeletalfibroproliferative disorders, especially Dupuytren's disease and to amethod of treating musculoskeletal fibroproliferative disorders,especially Dupuytren's disease.

BACKGROUND OF THE INVENTION

Dupuytren's disease, which is alternatively known as palmar fibromatosis(or in its established disease state Dupuytren's contracture), is adisease associated with the build up of extracellular matrix materialssuch as collagen on the connective tissue of the hand (the palmarfascia) causing it to thicken and shorten with the physical effect ofcausing the fingers to curl, most commonly the ring finger and littlefinger.

Dupuytren's disease affects approximately 5% of the white Caucasianpopulation. The commonest manifestation is progressive flexioncontracture of the digits of the hand, resulting in significantlycompromised function. It affects both males and females, but theincidence is higher in males.

The causes of Dupuytren's disease are not well understood and underlyingdisease is not currently curable.

Treatment of Dupuytren's disease has traditionally been invasivesurgical techniques. Primarily, the treatment has involved surgicalexcision of the offending tissue. In severe or recurrent disease, thesurgical excision may be combined with excision of the overlying palmarskin and resurfacing of the cutaneous defect with full-thickness skingraft. Surgery is typically followed by prolonged rehabilitation,usually lasting 3 months and complications have been reported in up to20% of cases. Such surgical correction is the mainstay treatment oflater stage disease when secondary changes to tendons and joints havedeveloped. A less invasive surgical intervention is needle fasciotomy inwhich the fibrous bands (contractures) in connective tissue are dividedusing the bevel of a needle.

Enzymatic cleavage of the affected tissue has been the focus ofdevelopment to reduce invasiveness associated with surgery and improverecovery time. This approach has led to trials of collagenase. Abacterial collagenase, Clostridial collagenase, has been granted FDAapproval as Xiaflex™ to Pfizer and Auxilium. U.S. Pat. No. RE39,941,U.S. Pat. No. 5,589,171 and U.S. Pat. No. 6,086,872 describe the use ofbacterial collagenase for the enzymatic cleavage of connective tissue inthe treatment of Dupuytren's disease. Bacterial collagenases suffer fromcertain disadvantages: for example lack non-selective cleaving ofvarious collagen materials including collagen type IV associated withblood vessels; and, in the case of Xiaflex™, possible allergic reactionsand potential immunogenicity; and administration may cause haemorrhagewhilst the prolonged activity of collagenase limits the dose that can beadministered locally due to risk of side effects as the drug disperses.

WO 2010/102202 describes a novel temperature sensitive recombinantcollagenase in which the activity is observed at significantly belowbody temperature, but which is comparatively inactive at bodytemperature. Thus Dupuytren's syndrome can be treated by administeringsuch recombinant collagenase at lower temperatures, which it is claimedrestricts the duration of activity, increases the possible local doseand reduces collagenase-related side effects.

To date collagenase therapies have appeared relatively effective intreatment of contracture of the metacarpophalangel joint, whilst thecorrection of proximal interphalangeal joints has been much lesssatisfactory. Furthermore, as with surgical interventions, recurrencecan be expected, but in the case of early collagenase trials, whichinvolve enzymatically cutting the cord, recurrence is high, especiallyfor disease affecting the proximal interphalangeal joint.

Other non-surgical treatments that have been proposed includeapplication of vitamin E cream applied as topical therapy, ultrasonictherapy and low-dose radiation therapy (for slowing the progression ofearly stage disease), such as X-rays and electron beam therapy.

Most research for treatments of Dupuytren's disease has focused ondetecting pre-disposition to Dupuytren's (e.g. US-A-2004/0161761) and onthe extracellular matrices produced, which has resulted in thecollagenase-based treatments. There has been very little conclusiveinsight into potential treatments gained from studies into thebiochemical pathway of Dupuytren's disease.

There remains a need for novel therapeutic intervention in the treatmentand/or prevention of (e.g. progression of) Dupuytren's disease and othermusculoskeletal fibroproliferative disorders.

The present inventors have found that administration of a TNF-αantagonist is surprisingly effective on its own or in combination withanother Dupuytren's treatment in preventing the progression of earlystage Dupuytren's disease and reversing later stage Dupuytren's diseaseas well as reducing recurrence of disease.

Problem to be Solved by the Invention

There remains a need for improvements in the treatment of Dupuytren'sdisease and other musculoskeletal fibroproliferative disorders,particularly fibromatosis and like diseases including and preferablyselected from plantar fibromatosis (or Ledderhose's disease), adhesivecapsulitis (frozen shoulder) and Peyronie's disease (fibromatosis of thepenis).

It is an object of this invention to provide a composition and methodfor the treatment or prophylaxis (e.g. prevention of progression orrecurrence) of one or more of Dupuytren's disease, plantar fibromatosis,adhesive capsulitis and Peyronie's disease.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention, there is provided acomposition for use in the treatment of a musculoskeletalfibroproliferative disorder, the composition comprising (e.g. atherapeutic, prophylatic or progression-inhibiting effective amount of)a TNF-α antagonist.

In a second aspect of the invention, there is provided a TNF-αantagonist for use in the treatment of a musculoskeletalfibroproliferative disorder. There is also provided the use of a TNF-αantagonist in the manufacture of a medicament for the treatment of amusculoskeletal fibroproliferative disorder.

In a third aspect of the invention, there is provided a composition foruse in the treatment of a musculoskeletal fibroproliferative disorder,the composition comprising (e.g. a therapeutic, prophylactic orprogression-inhibiting effective amount of) a DAMP antagonist and/or anAGE inhibitor.

In a fourth aspect of the invention, there is provided use of a DAMPantagonist and/or an AGE inhibitor in the manufacture of a medicamentfor the treatment of a musculoskeletal fibroproliferative disorder.

In a fifth aspect of the invention, there is provided a composition foruse in the treatment of a musculoskeletal fibroproliferative disorder,the composition comprising (e.g. a therapeutic, prophylactic orprogression-inhibiting effective amount of) a DAMP and/or AGEinflammatory pathway inhibitor.

In a sixth aspect of the invention, there is provided use of a DAMPand/or AGE inflammatory pathway inhibitor in the manufacture of amedicament for the treatment of a musculoskeletal fibroproliferativedisorder.

In a seventh aspect of the invention, there is provided a method for thetreatment of a musculoskeletal fibroproliferative disorder, the methodcomprising administering to a patient in need thereof an effectiveamount of one or more of a DAMP antagonist, an AGE inhibitor or a DAMPand/or AGE inflammatory pathway inhibitor, alone or in combination withan extracellular matrix degradation, depletion or cleavage agent.

In an eighth aspect of the invention, there is provided a method for thetreatment of a musculoskeletal fibroproliferative disorder, the methodcomprising administering to a patient in need thereof an effectiveamount of a myofibroblast activity down-regulating agent and/or amyofibroblast production inhibitor, such as a TNF-α antagonist, alone orin combination with an extracellular matrix degradation, depletion orcleavage agent.

In a ninth aspect of the invention, there is provided a method forreduction or prevention of recurrence of Dupuytren's diseasepost-surgical fasciectomy, post-needle fasciotomy orpost-enzyme-mediated extracellular matrix degradation, the methodcomprising locally administering to a patient a myofibroblast activitydown-regulating agent and/or a myofibroblast production inhibitor.

Advantages of the Invention

The compositions and methods of the present invention enable progressionof Dupuytren's (and other fibromatosis and like disease) to be slowed orhalted. It has particular advantages in that early disease stateDupuytren's (and other fibromatosis and like disease) can be preventedfrom progressing to an established state disease and avoid surgicalintervention and the associated recovery time.

Compositions and methods of the present invention enable the treatment,prevention and inhibition of progression of musculoskeletal adhesionssuch as adhesive capsulitis and tendon adhesion (such as adhesion of theproximal interphalangeal joint in established disease state Dupuytren'sdisease).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images of nodules and cord in an intraoperative view;

FIG. 2 is a chart showing a distribution of α-SMA rich cells in tissueexcised from different parts of diseased Dupuytren's tissue;

FIG. 3 is a photograph of a Culture Force Monitor used in in vitroexperiments to assess contractile behaviour of cells in athree-dimensional collagen matrix;

FIG. 4 shows graphs of contraction versus time for different cellcultures (in a Culture Force Monitor of FIG. 3) over a 24 hour period;

FIG. 5 is a chart showing the mean rate of contraction for cells ofdifferent tissue derivation from Dupuytren's patients;

FIG. 6 charts mean rate of contraction for cells from different tissuefrom Dupuytren's patients, the amount of messenger RNA, amount andintracellular distribution of the contractile protein α-smooth muscleactin (α-SMA);

FIG. 7 shows images of inflammatory cells (macrophages, CD68, mastcells, mast cell tryptase) in Dupuytren's nodule and cord;

FIG. 8 shows images of sections of Dupuytren's cord samples stained forα-SMA and RAGE;

FIG. 9 shows images of sections of skin samples from Dupuytren'spatients stained for RAGE and showing differential distribution innon-palmar and palmar skin;

FIG. 10 provides charts showing FACS analysis for cells deriving fromnodular, non-palmar and palmar skin fibroblasts for expression of RAGE;

FIG. 11 provides charts showing FACS analysis for cells deriving frommatched sets of non-palmar and palmar skin for expression of RAGE;

FIG. 12 provides a chart of contractility for palmar skin dermalfibroblasts treated with TNF-α, HMGB1 or TGF-β1;

FIG. 13 is a chart showing the contractility of primary passagenodule-derived cells (from a Dupuytren's patient) and in the presence orabsence of anti-TNF-α.

FIG. 14 is a chart showing the contractility of palmar dermalfibroblasts exposed to AGEs

FIG. 15 is a chart showing TNF-α production from human monocytes exposedto certain DAMPs. LPS is a PAMP, shown as positive control.

FIG. 16 is a chart showing TNF-α production from human monocytes exposedto certain DAMPs in the presence of certain receptor blockers.

FIG. 17 is a chart showing TNF-α production from murine bone marrowcells in the presence of S100 A8 in turn in cells having TLR-4deficiency and MyD88 deficiency.

FIG. 18 is a chart showing TNF-α production from human monocytes exposedto a certain DAMP alone and in combination with LPS.

FIG. 19 is a schematic of proposed mechanism of the role of trauma andAlarmins in the pathogenesis of Dupuytren's disease.

FIG. 20 is a chart of fold induction in contraction of palmarfibroblasts on exposure to supernatant from monocytes stimulated withAGEs, with or without anti-TNF-α.

FIG. 21 is a chart showing the contractility of palmar dermalfibroblasts and dose response to TGF-β1.

FIG. 22 is a chart showing that palamar dermal fibroblasts from patientswith Dupuytren's disease exposed to TNF-α become more contractilewhereas non-palmar dermal fibroblasts do not.

FIG. 23 is a chart showing a dose related inhibition of contractility ofcells form Dupuytren's nodules exposed to TNF-α antagonist.

FIG. 24 is an image showing cells from a Dupuytren's nodule in a3-dimensional collagen gel exposed only to control IgG antibody stainedwith phalloidin and exhibiting alignment in axis of stress.

FIGS. 24 b and 24 c are images showing cells from a Dupuytren's nodulestained with phalloidin and α-SMA respectively, treated with a TNF-αantagonist, showing loss of alignment in axis of stress.

FIG. 25 is a schematic of a proposed role of advanced glycation endproducts, injury and alarmins in the pathogenesis of fibroproliferativedisorders, highlighting the key role of TNF-α in the final commonpathway.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for an improved treatment of a musculoskeletalfibroproliferative disorder, especially Dupuytren's disease (or otherfibromatosis and like disease such as plantar fibromatosis, adhesivecapsulitis and Peyronie's disease), which comprises administration to apatient in need thereof, especially a patient showing signs of earlydisease state, a therapeutic, prophylactic or progression-inhibitiveamount of a myofibroblast activity down-regulating agent and/or amyofibroblast production inhibitor and preferably comprisesadministration to the patient a therapeutic, prophylactic orprogression-inhibitive amount of a TNF-α antagonist. Further, theinvention provides, by administration of a TNF-α antagonist to a patienthaving or showing signs of developing Dupuytren's disease, prevention ofdisease manifestation and/or progression, optionally as an adjunctive(or concomitant) therapy to a primary surgical intervention (e.g. afasciotomy or fasciectomy) or primary therapeutic treatment (e.g. anextracellular matrix degradation, depletion or cleaving agent, such as amatrix metalloproteinase or collagenase). Still further, the inventionprovides, by administration of a TNF-α antagonist to a patient,prevention of recurrence of disease as an adjunctive therapy to primarysurgical intervention or therapeutic treatment of established disease.

Musculoskeletal fibroproliferative disorders are characterized byexcessive or uncontrolled production of extracellular matrix inassociation with a musculoskeletal structure, often associated withcontraction in later stage disease. As mentioned above, musculoskeletalfibroproliferative disorders include fibromatosis disorders. (The terms‘musculoskeletal fibroproliferative disorders’ and ‘fibromatosisdisease’ may be used interchangeably herein, where the context allows).The present invention is concerned with the treatment and, inparticular, the inhibition of progression and recurrence (e.g. afterprimary treatment by surgery or therapy) of such diseases. Inparticular, the present invention is concerned with diseases selectedfrom Dupuytren's disease, plantar fibromatosis, adhesive capsulitis andPeyronie's disease, especially Dupuytren's disease. The remainder ofthis document will discuss compositions and methods for treatment ofmusculoskeletal fibroproliferative disorders generally, with specificreference to Dupuytren's disease. Where the context allows, it should beunderstood that the disclosure may be read also with the generality orother specified diseases in place of Dupuytren's disease.

It is believed that the effectiveness of TNF-α antagonists in thetreatments of the present invention is due to the dependence on TNF-α ofdifferentiation of fibroblasts into myofibroblasts, which are understoodto be the main culprits in contractile activity and induction ofuncontrolled extracellular matrix generation in Dupuytren's disease (andother fibromatosis diseases). The inventors have demonstrated this TNF-αdependence and has identified antagonists of TNF-α as viabletherapeutics (contrary to the teaching of Goldberg et al, J InvestDermatol. 2007 November; 127(11): 2645-2655, which showed TNF-αsuppression of myofibroblast differentiation).

The clinical consensus is currently that clinical nodules are theprecursor to established Dupuytren's disease. Dupuytren's disease occursin people with genetic predisposition and further risk factors tomanifestation of Dupuytren's disease include local trauma, poorlifestyle (e.g. smoking and drinking alcohol and poor diet), liverdisease and diabetes. Established disease presents as flexioncontracture which may typically be presented as contracture of themetacarbophalangeal joints (MCPJ) alone, less frequently contracture ofthe proximal interphalangeal joints (PIPJ) alone, and often both. Aphase III clinical trial of enzymatic fasciotomy using bacterialcollagenase reported (Hurst et al, N. Engl J. Med, 2009, 361, 968-979)that 77% of MCPJ contractures were effectively treated (to within 5° offull extension) compared with 40% of PIPJ contractures. An earlier stagetrial (Badalamente et al, J Hand Surg Am, 2007, 32, 767-774) showedrecurrence rates of 57% in patients with PIPJ contractures at 2 yearsfollow-up.

Numerous studies have shown that the presence of myofibroblasts isconcomitant with early and active disease and that such cells areimplicated in proliferative extra-cellular matrix (ECM) generation ordeposition and, in particular, collagen deposition. TGF-β1 leads to thedevelopment of the myofibroblast phenotype. Myofibroblasts are alsobelieved to be responsible for contractile behavior. Myofibroblastscharacteristically express α-smooth muscle actin (α-SMA), which is theactin isoform typical of vascular smooth muscle cells. α-SMA is believedto be the protein responsible for the contractility of myofibroblastsand is the most reliable marker for myofibroblasts.

As mentioned above, the present invention preferably comprises acomposition and method for treating, and more preferably inhibiting orhalting the progression or recurrence of, musculoskeletalfibroproliferative disorders, such as fibromatosis disease, especiallyDupuytren's disease, by administering to a patient a therapeutic,prophylactic or progression-inhibiting amount of a TNF-α antagonist.Preferably, the administration is local administration (e.g. byinjection into or adjacent to the affected tissue).

There are two main embodiments of this invention.

A first main embodiment of the invention comprises a composition andmethod for treating early disease state musculoskeletalfibroproliferative disorders, especially early disease state Dupuytren'sdisease, by administering to a patient presenting early state disease,e.g. prior to the presence of palpable cord, an effective amount of aTNF-α antagonist.

According to the first embodiment, a composition comprising a TNF-αantagonist may be administered to a patient for preventing diseaseprogression (to established disease state) and resultant flexioncontracture. Preferably, the method comprise local administration (e.g.by injection) directly into the clinical nodule(s). In a preferredembodiment, the method further comprises administering to the patient,preferably locally (and more preferably directly to the clinicalnodule(s) identified), an extracellular matrix degradation, depletion orcleavage agent, which is preferably a collagen degradation, depletion orcleavage agent and may be, for example a matrix metalloproteinase (MMP)and/or a collagenase (but may be, for example, a MMP or collagenaseup-regulating or inducing agent). It is believed that the matrixmetalloproteinase or collagenase may disrupt collagen and extra-cellularmatrix local to the clinical nodule(s) thereby enhancing access ofadministered TNF-α antagonist to the proliferative fibrotic foci andthus enhance efficacy of treatment. It is believed that administrationof the TNF-α antagonist in this manner may be considered prophylactic orprogression halting or inhibiting treatment. According to thisembodiment, the primary treatment is the TNF-α antagonist to which theextracellular matrix degradation or cleavage agent is preferablyadjunctive.

In a preferred embodiment which involves the combined treatment of apatient presenting early disease state musculoskeletalfibroproliferative disorders, especially Dupuytren's disease, with aTNF-α antagonist and an extracellular matrix degradation, depletion orcleavage agent (e.g. matrix metalloproteinase and/or collagenase), theTNF-α antagonist and the extracellular matrix degradation, depletion orcleavage agent (e.g. collagenase) may be administered simultaneously orsequentially, together or separately. Preferably, both TNF-α antagonistand the extracellular matrix degradation, depletion or cleavage agent(e.g. collagenase) are administered locally, for example by injection.Optionally, they may be administered simultaneously, e.g. administeringa composition comprising both TNF-α antagonist and collagenase (e.g. byinjectable solution) or by applying two separate compositions at thesame time. Alternatively, the TNF-α antagonist and the extracellularmatrix degradation, depletion or cleavage agent (e.g. collagenase) areadministered separately. When administered separately, they may beadministered in any order a suitable time apart. Preferably, whenadministered separately the extracellular matrix degradation, depletionor cleavage agent (e.g. collagenase) is administered first followed bythe TNF-α antagonist, which may be administered a suitable time afterthe TNF-α antagonist, e.g. after no less than 5 minutes, and preferablywithin 48 hours, more preferably within 24 hours, still more preferablywithin 6 hours and most preferably within 15 minutes to 3 hours.

Preferably, the TNF-α antagonist and the extracellular matrixdegradation, depletion or cleavage agent are administered simultaneouslyfor the treatment of early disease state musculoskeletalfibroproliferative disorders. Preferably, a composition is provided forlocal administration (e.g. injectable solution, sustained releasecomposition or implant) for treating early disease state musculoskeletalfibroproliferative disorders, preferably Dupuytren's disease, whichcomposition comprises an effective amount of a TNF-α antagonist (orconfigured to release an effective amount of TNF-α antagonist if, forexample, the composition is a sustained release composition) optionallyin combination with an extracellular matrix degradation, depletion orcleavage agent (preferably a matrix metalloproteinase and/orcollagenase) preferably in an adjunctive amount and a pharmaceuticallyacceptable carrier.

Preferably, according to this embodiment, the TNF-α antagonist isprovided in an amount effective to inhibit disease progression withoutinducing systemic complications. Optionally, therefore, the TNF-αantagonist is provided in an amount to reduce myofibroblast activity inclinical nodule tissue by at least 10%, preferably at least 30%, morepreferably at least 50%, still more preferably at least 75% and mostpreferably at least 90%, as indicated, for example by, an averageα-SMA-positive myofibroblast cell population in clinical nodule tissueby at least 10%, preferably at least 30%, more preferably at least 50%,still more preferably at least 75% and most preferably at least 90%,which activity reduction or cell population reduction is preferablyobservable within 48 h, more preferably 24 h, from administration.Preferably, an effective amount of TNF-α antagonist is that which willresult in a reduction in clinical nodule size (e.g. at least a 20%, oreven at least a 50%, reduction in size, as measured by degree ofprotrusion or lateral or longitudinal extent) in up to two weeks postadministration. Efficacy of TNF-α antagonist treatment preferably isobservable by an overall reduction in the progression of disease.

Preferably the TNF-α antagonist may be administered in an amount that isin the range 0.01 to 0.5 of the dose indicated (or would be indicated)for systemic treatment of Rheumatoid Arthritis (e.g. by reference toMarketing Authorisation or FDA approval), preferably 0.05 to 0.2 andmore preferably 0.095 to 0.15 of the dose. Preferably, the TNF-αantagonist is selected from one or a combination of Infliximab,Adalimumab, Certolizumab pegol, Golimumab or Etanercept and mostpreferably the TNF-α antagonist is Certolizumab pegol, which ispreferably administered in an amount from 1 to 100 mg, preferably 5 to50 mg and most preferably 10 to 40 mg, e.g. as an injection into theclinical nodule(s). Where more than one injection is provided (e.g. totwo distinct clinical nodules), the dose is preferably divided so thetotal dose provided is in the above range.

Preferably, according to this embodiment, an extracellular matrixdegradation, depletion or cleavage agent, e.g. a matrixmetalloproteinase and/or collagenase, is provided in a TNF-α antagonistadjunctive amount, by which it is meant an amount effective to enhancethe efficacy of the TNF-α antagonist. In any case, it is preferred thatthe extracellular matrix degradation, depletion or cleavage agent (e.g.matrix metalloproteinase or collagenase) is provided in an amount of upto 1 mg. Preferably, the extracellular matrix degradation, depletion orcleavage agent (e.g. matrix metalloproteinase or collagenase) isadministered in an amount significantly below (e.g. 0.01 to 0.5 times)the extracellular matrix degradation, depletion or cleavage agent (e.g.matrix metalloproteinase or collagenase) dose that would be required toachieve an enzymatic fasciotomy in established disease statefibromatosis. Preferably, the extracellular matrix degradation,depletion or cleavage agent (e.g. matrix metalloproteinase orcollagenase) is provided in an amount of 0.01 to 0.5 mg, more preferably0.05 to 0.2 mg. The extracellular matrix degradation, depletion orcleavage agent, e.g. matrix metalloproteinase or collagenase, may assistthe TNF-α antagonist in accessing the cell mass, as well as assisting indisaggregating of the extracellular matrix of the clinical nodule.

A second main embodiment of the invention comprises a composition andmethod for treating established disease state Dupuytren's (or othermusculoskeletal fibroproliferative) disease by administering to apatient an effective amount of a TNF-α antagonist, preferably incombination with, simultaneous to, sequentially with, in associationwith, concomitantly with, in combined administration with or adjunctiveto surgical fasciectomy, a fasciotomy and/or a extracellular matrixdegradation, depletion or cleavage agent (e.g. a matrixmetalloproteinase or collagenase) treatment, preferably a collagenasetreatment. Preferably, the method comprises surgical fasciectomy, needlefasciotomy or extracellular matrix degradation, depletion or cleavageagent (e.g. a matrix metalloproteinase or a collagenase) administration,which provides improvement (i.e. enabling greater extension of theaffected digits), more preferably correction (i.e. to within 5° of fullextension) and most preferably full correction (complete extension) ofthe established disease. Most preferably, the method comprisesadministration of extracellular matrix degradation, depletion orcleavage agent (e.g. a matrix metalloproteinase or a collagenase) tosites local to the disease site. Where the treatment comprisesextracellular matrix degradation, depletion or cleavage agent (e.g. amatrix metalloproteinase or a collagenase), the TNF-α antagonist may beprovided for combined treatment by simultaneous, sequential or separateadministration, e.g. for combined, concomitant or adjunctive therapy.Preferably, in this embodiment, the TNF-α antagonist is adjunctive tothe extracellular matrix degradation, depletion or cleavage agent (e.g.a matrix metalloproteinase or a collagenase) treatment. According tothis embodiment, the TNF-α antagonist may be administered separately,before or after, administration of the extracellular matrix degradation,depletion or cleavage agent (e.g. a matrix metalloproteinase or acollagenase), e.g. up to 4 to 6 weeks before or after administration ofthe extracellular matrix degradation, depletion or cleavage agent (e.g.a matrix metalloproteinase or a collagenase), preferably up to 14 daysbefore or after administration of the extracellular matrix degradation,depletion or cleavage agent (e.g. a matrix metalloproteinase or acollagenase), still more preferably at least 30 minutes before or afteradministration of the extracellular matrix degradation, depletion orcleavage agent (e.g. a matrix metalloproteinase or a collagenase) andmore preferably after the extracellular matrix degradation, depletion orcleavage agent (e.g. a matrix metalloproteinase or a collagenase), e.g.in the period 4 hours to 7 days after the extracellular matrixdegradation, depletion or cleavage agent (e.g. a matrixmetalloproteinase or a collagenase), whereby administration of TNF-αantagonist may gain better access to the disease site but beadministered at a point when myofibroblasts can be optimally inhibited.

Preferably, according to this embodiment, the TNF-α antagonist isprovided in an amount effective to inhibit disease recurrence withoutinducing systemic complications. Optionally, therefore, the TNF-αantagonist is provided in an amount to reduce myofibroblast activity incord tissue or clinical nodules by at least 10%, preferably at least30%, more preferably at least 50%, still more preferably at least 75%and most preferably at least 90%, compared with post-surgery orfasciotomy (needle or enzyme), as indicated, for example by, an averageα-SMA-positive myofibroblast cell population in cord tissue or clinicalnodules tissue by at least 10%, preferably at least 30%, more preferablyat least 50%, still more preferably at least 75% and most preferably atleast 90%, which activity reduction or cell population reduction ispreferably observable within 48 h, more preferably 24 h, fromadministration. Preferably, the recurrence inhibitory effect may beachieved by maintaining populations of α-SMA-positive myofibroblastcells in cord, histological nodule or clinical nodules below 50% oftotal cell population, preferably below 30% and most preferably below15%, within, for example, two weeks of administration.

Preferably, an effective amount of TNF-α antagonist is that which willprevent a palpable or as measured (e.g. by ultrasound scan) increase inclinical nodule presentation and/or in clinical nodule size (e.g. a 25%increase in size, as measured by degree of protrusion or lateral orlongitudinal extent) in up to two to four weeks post administration.Efficacy of TNF-α antagonist treatment preferably is observable by anoverall prevention in the recurrence of disease (e.g. established statedisease). Preferably, by administering an effective amount of TNF-αantagonist, re-establishment of established state disease manifested byflexion contracture can be managed or prevented, e.g. flexioncontracture maintained to 10° or less, more preferably 5° or lessfurther contraction compared with post-correction treatment extent,within a period after administration of the TNF-α antagonist, e.g. up to6 weeks, preferably up to 6 months. Optionally, repeat administrationsmay be provided in order to achieve this (e.g. two to four weekly).

Preferably the TNF-α antagonist may be administered in an amount that isin the range 0.01 to 0.5 of the dose indicated (or would be indicated)for systemic treatment of Rheumatoid Arthritis (e.g. by reference toMarketing Authorisation or FDA approval), preferably 0.05 to 0.2 andmore preferably 0.095 to 0.15 of the dose. Preferably, the TNF-αantagonist is selected from one or a combination of Infliximab,Adalimumab, Certolizumab pegol, Golimumab or Etanercept and mostpreferably the TNF-α antagonist is Certolizumab pegol, which TNF-αantagonist (e.g. Certolizumab pegol) is preferably administered in anamount from 0.1 to 100 mg (e.g. 0.1 or 0.5 to 10 or 20 mg), preferably 1to 100 mg, more preferably 5 to 50 mg and most preferably 10 to 40 mg,e.g. as an injection into the clinical nodule(s). Where more than oneinjection is provided (e.g. to two distant or distinct clinicalnodules), the dose is preferably divided so the total dose provided isin the above range. Further, it should be noted that the optimal dosemay vary according to the TNF-α antagonist used. The optimal dose ispreferably a dose which provides the maximum inhibitive effect (onmyofibroblast activity and production) for which the isotype antibody atthe same dose has no, minimal or acceptably small effect.

Preferably, according to this main embodiment of the invention, thetreatment comprises administration local to disease site of anextracellular matrix degradation, depletion or cleavage agent (e.g. amatrix metalloproteinase or a collagenase). The extracellular matrixdegradation, depletion or cleavage agent (e.g. a matrixmetalloproteinase or a collagenase) should be administered in an amountsufficient to enable improvement and/or correction of disease-associatedcontraction (e.g. to 5° or less of full extent in the case ofDupuytren's) within 24 or 48 hours of administration). Preferably, anextracellular matrix degradation, depletion or cleavage agent, such as acollagenase (e.g. Clostridium collagenase), is provided for localadministration in an amount of up to 10 mg administered in one or morelocations along each contracture, preferably from 0.1 to 5 mg peradministration and more preferably from 0.15 to 2 mg and most preferably0.5 to 1 mg.

In one embodiment in which extracellular matrix degradation, depletionor cleavage agent (e.g. a matrix metalloproteinase or a collagenase) isadministered for contracture improvement and TNF-α antagonistadministered for recurrence inhibition, there may be provided a singlecombined dose of the extracellular matrix degradation, depletion orcleavage agent (e.g. a matrix metalloproteinase or a collagenase) andTNF-α antagonist.

In one embodiment, an extracellular matrix degradation, depletion orcleavage agent may be administered (e.g. injected) into diseased cordtissue in an effective amount, whilst TNF-α antagonist may beadministered (e.g. injected) into clinical nodule(s) and/or cord tissuein a recurrence-inhibitory amount.

Any known TNF-α antagonist may be utilized in the implementation of theinvention, a broad variety of which are known and disclosed in the art.The TNF-α antagonist is preferably a human TNF-α antagonist. Optionally,the TNF-α antagonist may be an antibody, such as a monoclonal antibodyor fragment thereof; a chimeric monoclonal antibody (such as ahuman-murine chimeric monoclonal antibody); a fully human monoclonalantibody; a recombinant human monoclonal antibody; a humanized antibodyfragment; a soluble TNF-α antagonist, including small molecule TNF-αblocking agents such as thalidomide or analogues thereof or PDE-IVinhibitors; a TNF receptor or a TNF receptor fusion protein, e.g. asoluble p55 or p75 TNF receptor or TNF receptor fusion protein.

Optionally, the TNF-α antagonist is a functional fragment or fusionprotein comprising a functional fragment of a monoclonal antibody, e.g.of the types mentioned above, such as a Fab, F(ab′)₂, Fv and preferablyFab. Preferably a fragment is pegylated or encapsulated (e.g. forstability and/or sustained release).

Optionally, the TNF-α antagonist is provided as a bi-functional (orbi-specific) antibody or bi-functional (or bi-specific) antibodyfragment. The bi-functional TNF-α antagonist antibody or fragmentthereof may be, for example, an antibody, such as a monoclonal antibodyor fragment thereof, a chimeric monoclonal antibody (such as ahuman-murine chimeric monoclonal antibody), a fully human monoclonalantibody, a recombinant human monoclonal antibody, a humanized antibodyfragment. Where the TNF-α antagonist comprises a bi-functional antibodyfragment or portion, it is preferably a bi-functional F(ab′)₂ fragmentor divalent ScFv, e.g. a bi-specific tandem di-ScFv. In any case, thebi-functional (or bi-specific) antibody or fragment thereof may compriseas one variable domain (e.g. antigen binding portion) a TNF-α antagonist(e.g. a TNF-α antagonist portion of Infliximab, Adalimumab,Certolizumab, Golimumab or Etanercept) and as the other variable domain(e.g. antigen binding portion) a second variable domain other than TNF-αantagonist. Optionally, the second variable domain may comprise anantibody mobility inhibitor, which may be, for example an extracellularmatrix, e.g. collagen, binder or antagonist. Thereby, a higher dose ofTNF-α antagonist may be administered since the antibody or fragmentthereof will be self-localising, minimizing systemic uptake and thussystemic side effects. Optionally, the second variable domain maycomprise a DAMP antagonist (such as an antagonist for S100A8 and/orS100A9, e.g. as described in U.S. Pat. No. 7,553,488) or an AGEinhibitor (e.g. being variable domains of DAMP antagonist antibody orAGE inhibitor antibody). Methods for the production of bi-functionalantibodies, and bi-functional antibody fragments are known in the art,which methods may be applied to the present purpose. Preferably, theTNF-α antagonist is selected from those which at administration (e.g.local administration, such as injection into clinical nodule or cord)cause administration-site irritation manifested as palpable localswelling, redness and pruritis in fewer than 40% of patients, preferablyfewer than 20% and more preferably fewer than 10%.

The TNF-α antagonist may be selected, for example, from one or acombination of Infliximab, Adalimumab, Certolizumab pegol, Golimumab orEtanercept, or functional fragment thereof. Most preferably, the TNF-αantagonist is Certolizumab pegol, since it causes low injection sitereaction and pain.

It is particularly advantageous according to the present invention tominimize inflammation, irritation and pain associated withadministration since local irritation may limit patient acceptabilityand furthermore local inflammation may lead to recurrence of disease. Inone embodiment, the TNF-α antagonist may be administered with or priorto an extracellular matrix (ECM) degradation or cleavage agent (e.g.collagenase) whereby the inflammatory response to ECM degradation may beminimised, thereby reducing the likelihood of treatment inducedrecurrence.

The extracellular matrix (ECM) degradation, depletion or cleavage agentmay be any suitable agent capable of degrading, cleaving or causing orinducing degradation or cleavage of extracellular matrix, includingfibronectin and collagen. For example, the ECM degradation or cleavageagent may be an ECM degradation enzyme or an ECM degradation enzymeexpression up-regulator (e.g. relaxin). Preferably the ECM degradationor cleavage agent is a matrix metalloproteinase or a collagenase, morepreferably a collagenase, such as a bacterial collagenase (e.g.clostridial collagenase), human or humanised collagenase or mutant orrecombinant collagenase or recombinant matrix metalloproteinase (e.g.recombinant matrix metalloproteinase I, preferably human recombinantmatrix metalloproteinase I). Preferably, the collagenase is time ortemperature dependent or is photodynamically activated or deactivated,to allow higher local doses to be administered without systemic orlong-lasting side-effects. Optionally, it is a Cathespin-L or a mutantor recombinant thereof. Examples of suitable collagenase for use in thepresent invention include those described in: GB-A-2323530, U.S. Pat.No. 5,589,171, U.S. Pat. No. RE39,941, U.S. Pat. No. 6,086,272 &WO-A-2010/102262 (and for established disease optionally in the amountsdescribed therein, the disclosure of which collagenases and amounts andmodes of administration are incorporated herein by reference).

By early disease state it is meant that indications of disease arepresent, e.g. histological markers or more particularly clinical nodulesin tissue, but in the absence of, for example, palpable cord orsignificant contracture. By early disease state Dupuytren's disease, itis meant that indications of Dupuytren's disease are present, e.g.histological markers or more particularly clinical nodules in palmarand/or digital tissue, but in the absence of significant (e.g. at least5°) flexion contracture (or, for example, palpable cord).

By established disease state, it is meant that clinical nodules arepresent, palpable cord is present and contracture is evident. Byestablished disease state Dupuytren's disease, it is meant that clinicalnodules are present on the palm and digits of the hand and flexioncontracture is evident (e.g. at least 5°).

Varying histological stages of Dupuytren's disease have been categorisedin the literature, most succinctly by Rombouts (J Hand Surg Am, 14,644-652, 1989) and later authors, into three distinct stages: 1) aproliferative stage with high cellularity and the presence of mitoticfigures; 2) a fibrocellular stage charactised by high cellularity but nomitotic figures and the presence of reticulin network; and 3) a fibrousstage with few cells separated by broad bundles of collagen fibres.Stage 1) disease is believed to correlate with early disease state asdiscussed above (i.e. presence of nodules but no contracture) andDupuytren's stages 2) and 3) is believed to correlate with ourEstablished Disease State (characterized by digital contracture). Thepresent inventors have found that during early established diseasestate, active myofibroblasts are collected in the established nodulesand cords, especially in relation to the MCP and PIP joints and thesedrive the progression of flexion contractures of the digit.

By clinical nodule, it is meant a palmar or digital nodule evident as apalpable subcutaneous lump.

By histological (or histopathological) nodule, it is meant a collectionof cells (mainly myofibroblast cells with some inflammatory cells suchas macrophages and mast cells) typically in a whorled pattern and whichmay range from tiny foci of cells to larger collections of cells, butnot clinically palpable.

Without being bound by theory, it is believed that the initialclinically palpable nodule(s) is the focus of proliferating fibroblastsin disease progression, but that numerous histological nodules will format various locations in the palm and/or digits which will ultimatelycontribute to cord formation, contraction and flexion contracture.

Where ‘nodule’ is used herein it may be clinical or histological nodules(or either) as will be apparent from the context.

According to two alternative embodiments specific to Dupuytren's, afirst embodiment may relate to a composition and method for treatingDupuytren's disease characterized by joint contractures of less than 20°and a second embodiment may relate to a composition and method fortreating Dupuytren's disease characterized by joint contractures of atleast 20°. The contracture of 20° is identified as a transition phase,since at less than 20° contracture, many patients may choose to stopprogression of the disease without wishing to undergo surgery sincetheir mobility and operative use of the hand is still largely adequate,whilst at greater than 20°, many patients will find surgery or othercollagen depleting therapy (such enzymatic fasciotomy) essential torestore full function to the hand.

The inventors' investigations reveal that TNF-α as an optimaltherapeutic target for early Dupuyten's disease (i.e. early diseasestate). In established disease state Dupuytren's disease, an idealcombination is a matrix metalloproteinase such as collagenase with aTNF-α antagonist to inhibit recurrence, which is typically associatedwith enzymatic fasciotomy.

As mentioned above, myofibroblasts are implicated in two ways in thedevelopment of musceloskeletal fibroproliferative disorders and, inparticular, Dupuytren's disease. They are responsible for extracellularmatrix production or deposition and contractile behavior. It is believedthat the activity of myofibroblasts is mediated by α-SMA, which isover-expressed in active myofibroblast cells. Without being bound bytheory, the present inventors have found that TNF-α is implicated in theactivity of myofibroblasts in Dupuytren's disease in at least twoways—firstly, by reducing the activity of myofibroblast; and secondly byenhancing the production or attraction of myofibroblasts.

In each of the embodiments, the TNF-α antagonist may be provided in amultiple administrations over an extended (or continuous) term in orderto prevent or inhibit disease progression or recurrence. Whererecurrence is to be avoided, intermittent treatment may be provided by,e.g. low-dose fortnightly, monthly or six-monthly administration.Alternatively, continuous treatment may be provided by low-dosereleasing sustained or delayed intermittent release implant or patch.Alternatively, repeat doses may be initiated by signs of diseaseprogression in the early disease state and may optionally comprise acombined extracellular matrix degradation or cleavage agent (e.g. amatrix metalloproteinase or a collagenase) and TNF-α antagonisttreatment (e.g. consistent with the first embodiment described above).

In one embodiment of the invention, the progression of early diseasestate disease (e.g. Dupuytren's disease) to established disease statecan be prevented, inhibited or halted by the local administration of aTNF-α antagonist.

Preferably, the TNF-α antagonist may be administered separately orsimultaneously in combination with or adjunctively to a collagenaseand/or matrix metalloproteinase. A collagenase, especially aphoto-responsive or temperature dependent collagenase, may beadministered for local effect to enhance the TNF-α antagonist diseaseprogression inhibition effect by enhancing access to treatment sites bycleaving early stage extracellular matrix formation. A temperaturedependent collagenase is one which (typically a recombinant or mutantcollagenase) has collagenase activity dependent upon temperature andtypically is active at below body temperature, e.g. at 25° C. and below,thereby allowing extremely high doses of collagenase to act very locally(e.g. by injecting at the disease site at say 20° C. without having anysystemic action or other side effects associated with longevity ofaction). TNF-α antagonist have a further beneficial effect since it willreduce inflammation at the nodule site and thus reduce development andrecruitment of further myofibroblasts.

The composition and method of the present invention may utilise anysuitable means of administration, which is preferably local. Inparticular, the TNF-α antagonist should be administered locally, e.g. byapplying directly into a surgical incision during surgery, by injection(preferably directly into the clinical nodule(s) and/or cord tissue), byrelease from a sustained and/or delayed release lozenge or device thatmay be implanted into or close to the disease site or a sustained and/ordelayed release patch formulation, by topical application or any othersuitable route. A composition is preferably suitably formulated andtypically comprises the required dose of TNF-α antagonist along with apharmaceutical acceptable carrier or excipient.

Formulations for parenteral administration may typically comprise asterile aqueous preparation of the active ingredient, which ispreferably isotonic with the blood of the recipient. Formulations forintra-articular administration may be in the form of a sterile aqueouspreparation of the active ingredient. Formulations suitable for topicaladministration may include liquid and semi liquid preparations such asliniments, lotions and applications; oil-in-water and water-in-oilemulsions such as creams, ointments and pastes; and solutions andsuspensions.

In a further aspect, there is provided a formulation for frequent, e.g.daily, periodic or occasional (preferably daily), topical application tothe musculoskeletal fibroproliferative disorder area (e.g. the hands,and in particular palms and digits, in the case of Dupuytren's disease)for use, for example, by early disease state or post-operative patientsfor the inhibition of disease progression or recurrence, the formulationcomprising a TNF-α antagonist suitable for topical administration (e.g.selected from such TNF-α antagonists defined above) and a suitableexcipient. The formulation may be provided as a cream or lotion, a patchor a medicated glove (in which the glove is impregnated for release ofthe active component from the internal surface). Preferably, theformulation comprises TNF-α antagonist in a concentration foradministration by topical application of a low dose, such as 0.001 to0.05, preferably 0.001 to 0.01, of the systemic dose of the TNF-αantagonist. Optionally, the formulation further comprises a DAMPantagonist and/or an AGE inhibitor.

Optionally, the compositions and methods of the present invention mayfurther comprise further active ingredients that may be effective in thetreatment or progression-inhibition of musculoskeletalfibroproliferative disorders such as Dupuytren's disease. For example,combination therapy or concomitant or adjunctive co-administration of aTNF-α antagonist and an agent of the vascular endothelial growth factorfamily, such as VEGF-A, VEGF-B, VEGF-C or VEGF-D or an agent encodingsaid VEGF or a functional fragment thereof (such as described inWO-A-2004/082705), which combination is preferably a developmentretarding combination (or composition) for use in association withsurgery, or needle or enzyme fasciotomy. Additionally or alternativelysuch method or composition as described herein may further comprise anactivator of PPARγ (such as pioglitazone) for reducing myofibroblastpopulations local to the disease site (and enhancing the TNF-αantagonist activity).

In the treatment of musculoskeletal fibroproliferative disorders and,preferably, Dupuytren's disease, there is as a further aspect provided acomposition for use in such treatment which comprises a matrixmetalloproteinase or collagenase (or matrix metalloproteinase orcollagenase up-regulator) in combination with a myofibroblast activitydown-regulator and/or a myofibroblast production (or differentiation)inhibitor each preferably in appropriate therapeutic amounts accordingto the respective embodiment as discussed above. The preferredmyofibroblast activity down-regulator and/or myofibroblast production(or differentiation) inhibitor is TNF-α antagonist.

In a further aspect there is provided a method for the reduction orprevention of recurrence of musculosekeletal fibroproliferativedisorder, especially Dupuytren's disease, which comprises, aftersurgical, needle fasciotomy or enzyme fasciotomy, administration of amyofibroblast de-activating and/or producing inhibition agent local tothe disease site. Preferably the agent is a TNF-α antagonist, preferablyadministered in the aforementioned doses (relative the second mainembodiment).

In a further aspect of the invention, there is provided a method andcomposition for the treatment of a musculoskeletal fibroproliferativedisorder, especially Dupuytren's disease, which comprises a DAMP (damageassociated molecular patterns) antagonist. The musculoskeletalfibroproliferative disorder may be a fibromatosis and may preferably beselected from plantar fibromatosis, adhesive capsulitis, Peyronie'sdisease or Dupuytren's disease and is preferably Dupuytren's disease.The method preferably comprises administering an effective amount of aDAMP antagonist locally to the disease site, e.g. by injection intoclinical nodules of early disease state tissue or into nodules and/orcord of established disease state tissue or by application of asustained release patch or implant or application of a cream (or othersuch topical formulation). The composition according to this aspectpreferably comprises a DAMP antagonist in an effective amount and apharmaceutically acceptable excipient.

There have been observed some evidence to support a higher incidence ofDupuytren's disease in manual workers and association between injury,surgery and infection and Dupuytren's disease has been observed. Withoutbeing bound by theory, it is believed that DAMPs and, in particular, asub-group of DAMPs, Alarmins released as a result of trauma to the hand(or local area which disease affects) catalyse or induce myofibroblastproduction and/or myofibroblast activity (including contraction),especially in those genetically predisposed to the disease. It isbelieved that the Alarmins directly and/or indirectly inducemyofibroblast activity and/or production by direct biochemical pathwayfor differentiation of fibroblasts to active myofibroblasts andindirectly by up-regulating TNF-α production (which itself promotesmyofibroblast activity and production direction and throughinflammation-induced stress as discussed above). The Alarmins includeHMGB-1 (high mobility group box protein), S100 A8, S100 A9, S100 A8/9and S100 A12 and most implicated is S100 A8. The DAMP antagonistaccording to the method and composition according to this aspect of theinvention is preferably an Alarmin antagonist and more preferably one ormore antagonist of one or more of HMGB-1, S100 A8, S100 A9, S100 A8/9and S100 A12, most preferably an S100 A8 antagonist, e.g. an inactivefragment of S100 A8 to act as S100 A8 receptor blocker. Without beingbound by theory, it is believed that S100 A8 (and other alarmins) elicittheir inflammatory effect by binding TLR 4 (toll like receptor 4) andthus TLR 4 blockers may be considered S100 A8 antagonists.

Preferably, the DAMP antagonists of this aspect are for use inprevention or inhibition of the onset and/or development of disease inpre-disease and early disease state patients, especially trauma-inducedearly disease state patients (especially of Dupuytren's disease).Alternatively or additionally, the DAMP antagonists of this aspect arefor use in prevention or inhibition of recurrence of disease inpost-treatment patients having established state disease (e.g. aftersurgery or needle or enzyme fasciotomy), e.g. adjunctive to such primarytreatment, which is particularly beneficial since the primary treatmentis at risk of causing trauma-induced DAMP up-regulation and release andassociated myofibroblast activation. Accordingly, the DAMP antagonist ispreferably used in an amount effective to prevent or inhibit diseaseonset, progression or recurrence without inducing systemiccomplications. Optionally, therefore, the DAMP antagonist is provided inan amount to reduce myofibroblast activity in cord tissue or clinicalnodules by at least 5%, preferably at least 10% and more preferably atleast 30% and optionally 50% or more, 75% or more or even 90% or more,compared with post-trauma or post-surgery or fasciotomy (needle orenzyme), as indicated, for example by, an average α-SMA-positivemyofibroblast cell population in cord tissue or clinical nodules tissueby at least 10%, preferably at least 30%, more preferably at least 50%,still more preferably at least 75% and most preferably at least 90%,which activity reduction or cell population reduction is preferablyobservable within 48 h, more preferably 24 h, from administration.Preferably, the recurrence inhibitory effect may be achieved bymaintaining populations of α-SMA-positive myofibroblast cells in cord,histological nodule or clinical nodules below 50% of total cellpopulation, preferably below 30% and most preferably below 15%, within,for example, two weeks of administration.

Preferably, an effective amount of DAMP antagonist is that which willprevent a palpable increase in clinical nodule presentation and/or inclinical nodule size (e.g. a 25% increase in size, as measured by degreeof protrusion or lateral or longitudinal extent) in up to two to fourweeks post administration. Efficacy of DAMP antagonist treatmentpreferably is observable by an overall prevention in the onset ordevelopment of disease in post-trauma patients or in overall preventionof recurrence of disease (e.g. established state disease) inpost-surgery patients. Preferably, by administering an effective amountof DAMP antagonist (after primary treatment and intermittently inresponse to local trauma), re-establishment of established state diseasemanifested by flexion contracture can be managed or prevented, e.g.flexion contracture maintained to 10° or less, more preferably 5° orless further contraction compared with post-correction treatment extent,within a period after administration of the DAMP antagonist, e.g. up to6 weeks, preferably up to 6 months. Optionally, repeat administrationsmay be provided in order to achieve this (e.g. two to four weekly orresponsive to local trauma).

Optionally, the DAMP antagonist of this aspect of the invention may beco-administered or administered in combination (e.g. simultaneously orsequentially) with a TNF-α antagonist whereby two pathways tomyofibroblast activation may be controlled. In one embodiment, there isprovided a composition for the treatment of a musculoskeletalfibroproliferative disorder such as Dupuytren's disease, which comprisesan effective combined amount of a DAMP antagonist and a TNF-α antagonistand a pharmaceutically acceptable excipient. The composition may beadministered by injection (or other application) to the disease site(e.g. clinical nodules or cord tissue) or by application of a patch orby delayed and/or sustained release for local administration (e.g.implant) or topical application.

Optionally, the actives and compositions of this aspect of the inventionmay be provided as combined, concomitant or adjunctive treatment with anextracellular matrix degradation, depletion or cleavage agent as and inthe manner hereinbefore described (e.g. in place of and/or in additionto TNF-α antagonist).

In another aspect of the invention there is provided a method for themodulation of myofibroblast activity by DAMP agonism/antagonism, e.g. byadministration of a DAMP agonist or antagonist. There is also provided acomposition for up-regulating myofibroblast activity comprising a DAMPagonist and a composition for down-regulation myofibroblast activitycomprising a DAMP antagonist. The DAMP agonist or antagonist beingprovided in a myofibroblast modulating amount, optionally formulated forlocal administration.

Optionally, the DAMP antagonist is provided as a bi-functional (orbi-specific) antibody or bi-functional (or bi-specific) antibodyfragment. The bi-functional DAMP antagonist antibody or fragment thereofmay be, for example, an antibody, such as a monoclonal antibody orfragment thereof, a chimeric monoclonal antibody (such as a human-murinechimeric monoclonal antibody), a fully human monoclonal antibody, arecombinant human monoclonal antibody, a humanized antibody fragment.Where the DAMP antagonist comprises a bi-functional antibody fragment orportion, it is preferably a bi-functional F(ab′)₂ fragment or divalentScFv, e.g. a bi-specific tandem di-ScFv. In any case, the bi-functional(or bi-specific) antibody or fragment thereof may comprise as onevariable domain (e.g. antigen binding portion) a DAMP antagonist (suchas an antagonist for S100A8 and/or S100A9, e.g. as described in U.S.Pat. No. 7,553,488) and as the other variable domain (e.g. antigenbinding portion) a second variable domain other than DAMP antagonist.Optionally, the second variable domain may comprise an antibody mobilityinhibitor, which may be, for example an extracellular matrix, e.g.collagen, binder or antagonist. Thereby, a higher dose of DAMPantagonist may be administered since the antibody or fragment thereofwill be self-localising, minimizing systemic uptake and thus systemicside effects. Optionally, the second variable domain may comprise an AGEinhibitor (e.g. being variable domains of DAMP antagonist antibody orAGE inhibitor antibody). Methods for the production of bi-functionalantibodies, and bi-functional antibody fragments are known in the art,which methods may be applied to the present purpose.

Optionally, treatment of a patient with DAMP antagonist, optionally incombination with TNF-α antagonist, may be indicated for patients withearly disease state (for inhibition of disease progression) orpost-surgery (for recurrence inhibition) for patients who disease area(e.g. hands in the case of Dupuytren's is subject to local trauma), suchas, in the case of Dupuytren's disease, golfers, builders or driversetc.

Optionally, e.g. for patient's for whom local trauma is a causativefactor in the musculoskeletal fibroproliferative disorder, especiallyDupuytren's disease, there is provided (as a further aspect) aformulation for frequent, e.g. daily, periodic or occasional (preferablydaily), topical application to the musculoskeletal fibroproliferativedisorder area (e.g. the hands, and in particular palms and digits, inthe case of Dupuytren's disease) for use, for example, by early diseasestate or post-operative patients for the inhibition of diseaseprogression or recurrence, the formulation comprising a DAMP antagonistsuitable for topical administration (e.g. an S100 A8 and/or A9antagonist) and a suitable excipient. The formulation may be provided asa cream or lotion, a patch or a medicated glove (in which the glove isimpregnated for release of the active component from the internalsurface). Preferably, the formulation comprises a DAMP antagonist in aconcentration for administration by topical application of a low dose,such as 0.001 to 0.05, preferably 0.001 to 0.01, of the systemic dose ofthe DAMP antagonist. Optionally, the formulation further comprises anAGE inhibitor.

In a still further aspect of the invention, there is provided a methodand composition for the treatment of a musculoskeletalfibroproliferative disorder, especially Dupuytren's disease, whichcomprises an AGE (advanced glycation end products) inhibitor. Themusculoskeletal fibroproliferative disorder may be a fibromatosis andmay preferably be selected from plantar fibromatosis, adhesivecapsulitis, Peyronie's disease or Dupuytren's disease and is preferablyDupuytren's disease. The method preferably comprises administering aneffective amount of an AGE inhibitor locally to the disease site, e.g.by injection into clinical nodules of early disease state tissue or intonodules and/or cord of established disease state tissue or byapplication of a sustained release patch or implant or application of acream (or other such topical formulation). The composition according tothis aspect preferably comprises an AGE inhibitor in an effective amountand a pharmaceutically acceptable excipient.

There is a statistically significant association of Dupuytren's diseasewith smoking, alcohol consumption and diabetes mellitus. The presentinventors propose that AGE may be implicated in the association andbiochemical pathway driving Dupuytren's disease in geneticallypre-disposed patients who smoke, drink or have diabetes. AGE-modifiedproteins are the final products formed from irreversible non-enzymaticglycation between reducing sugars and polypeptides and have been shownto exert their influence by forming protein cross-links that alterextracellular matrix structure as well as interacting with cell surfacereceptors. Without being bound by theory, it is believed that AGEs andtheir receptors RAGE are implicated in the early stages and developmentof Dupuytren's disease and that AGEs up-regulate myofibroblast activityboth direction and indirectly. It is believed that AGEs present inpre-disease or early disease palmar tissue present due to increasedlevels associated with lifestyle choices or diabetes catalyse or inducemyofibroblast production and/or myofibroblast activity (includingcontraction), especially in those genetically predisposed to thedisease. It is believed that the AGEs directly and/or indirectly inducemyofibroblast activity and/or production by direct biochemical pathwayfor differentiation of fibroblasts to active myofibroblasts andindirectly by up-regulating TNF-α production (which itself promotesmyofibroblast activity and production direction and throughinflammation-induced stress as discussed above).

Preferably, the AGE inhibitors of this aspect are for use in preventionor inhibition of the onset and/or development of disease in pre-diseaseand early disease state patients (especially of Dupuytren's disease).Alternatively or additionally, the AGE inhibitors of this aspect are foruse in prevention or inhibition of recurrence of disease inpost-treatment patients having established state disease (e.g. aftersurgery or needle or enzyme fasciotomy), e.g. adjunctive to such primarytreatment. Accordingly, the AGE inhibitor is preferably used in anamount effective to prevent or inhibit disease onset, progression orrecurrence without inducing systemic complications. Optionally,therefore, the AGE inhibitor is provided in an amount to reducemyofibroblast activity in cord tissue or clinical nodules by at least5%, preferably at least 10% and more preferably at least 30% andoptionally 50% or more, 75% or more or even 90% or more, compared withpost-surgery or fasciotomy (needle or enzyme), as indicated, for exampleby, an average α-SMA-positive myofibroblast cell population in cordtissue or clinical nodules tissue by at least 10%, preferably at least30%, more preferably at least 50%, still more preferably at least 75%and most preferably at least 90%, which activity reduction or cellpopulation reduction is preferably observable within 48 h, morepreferably 24 h, from administration. Preferably, the recurrenceinhibitory effect may be achieved by maintaining populations ofα-SMA-positive myofibroblast cells in cord, histological nodule orclinical nodules below 50% of total cell population, preferably below30% and most preferably below 15%, within, for example, two weeks ofadministration.

Preferably, an effective amount of AGE inhibitor (e.g. pimagedine) isthat which will prevent a palpable increase in clinical nodulepresentation and/or in clinical nodule size (e.g. a 25% increase insize, as measured by degree of protrusion or lateral or longitudinalextent) in up to two to four weeks post administration. Efficacy of AGEinhibitor treatment preferably is observable by an overall prevention inthe onset or development of disease in post-trauma patients or inoverall prevention of recurrence of disease (e.g. established statedisease) in post-surgery patients or prevention of progression of earlydisease. Preferably, by administering an effective amount of AGEinhibitor (after primary treatment), re-establishment of establishedstate disease manifested by flexion contracture can be managed orprevented, e.g. flexion contracture maintained to 10° or less, morepreferably 5° or less further contraction compared with post-correctiontreatment extent, within a period after administration of the AGEinhibitor, e.g. up to 6 weeks, preferably up to 6 months. Optionally,repeat administrations may be provided in order to achieve this (e.g.two to four weekly or responsive to local trauma).

Any suitable AGE inhibitor may be utilized according to this aspect ofthe invention.

Optionally, the AGE inhibitor of this aspect of the invention may beco-administered or administered in combination (e.g. simultaneously orsequentially) with one or both of a DAMP antagonist and a TNF-αantagonist whereby at least two pathways to myofibroblast activation maybe controlled. In one embodiment, there is provided a composition forthe treatment of a musculoskeletal fibroproliferative disorder such asDupuytren's disease, which comprises an effective combined amount of anAGE inhibitor and a DAMP antagonist and/or a TNF-α antagonist and apharmaceutically acceptable excipient. The composition may beadministered by injection (or other application) to the disease site(e.g. clinical nodules or cord tissue) or by application of a patch orby delayed and/or sustained release for local administration (e.g.implant) or topical application.

Optionally, the actives and compositions of this aspect of the inventionmay be provided as combined, concomitant or adjunctive treatment with anextracellular matrix degradation, depletion or cleavage agent as and inthe manner hereinbefore described.

Optionally, the AGE inhibitor is provided as a bi-functional (orbi-specific) antibody or bi-functional (or bi-specific) antibodyfragment. The bi-functional AGE inhibitor antibody or fragment thereofmay be, for example, an antibody, such as a monoclonal antibody orfragment thereof, a chimeric monoclonal antibody (such as a human-murinechimeric monoclonal antibody), a fully human monoclonal antibody, arecombinant human monoclonal antibody, a humanized antibody fragment.Where the AGE inhibitor comprises a bi-functional antibody fragment orportion, it is preferably a bi-functional F(ab′)₂ fragment or divalentScFv, e.g. a bi-specific tandem di-ScFv. In any case, the bi-functional(or bi-specific) antibody or fragment thereof may comprise as onevariable domain (e.g. antigen binding portion) an AGE inhibitor and asthe other variable domain (e.g. antigen binding portion) a secondvariable domain other than AGE inhibitor. Optionally, the secondvariable domain may comprise an antibody mobility inhibitor, which maybe, for example an extracellular matrix, e.g. collagen, binder orantagonist. Thereby, a higher dose of AGE inhibitor may be administeredsince the antibody or fragment thereof will be self-localising,minimizing systemic uptake and thus systemic side effects. Methods forthe production of bi-functional antibodies, and bi-functional antibodyfragments are known in the art, which methods may be applied to thepresent purpose.

Optionally, treatment of a patient with an AGE inhibitor, optionally incombination with TNF-α antagonist, may be indicated for patients withearly disease state (for inhibition of disease progression) orpost-surgery (for recurrence inhibition) for patients who are diabeticor who smoke or drink (greater than the WHO recommended amount).

Optionally, e.g. for patients for whom diabetes or lifestyle (e.g.smoking and/or drinking excessive alcohol) is considered a causativefactor in the musculoskeletal fibroproliferative disorder, especiallyDupuytren's disease, there is provided (as a further aspect) aformulation for frequent, e.g. daily, periodic or occasional (preferablydaily), topical application to the musculoskeletal fibroproliferativedisorder area (e.g. the hands, and in particular palms and digits, inthe case of Dupuytren's disease) for use, for example, by early diseasestate or post-operative patients for the inhibition of diseaseprogression or recurrence, the formulation comprising an AGE inhibitorsuitable for topical administration and a suitable excipient. Theformulation may be provided as a cream or lotion, a patch or a medicatedglove (in which the glove is impregnated for release of the activecomponent from the internal surface). Preferably, the formulationcomprises an AGE inhibitor in a concentration for administration bytopical application of a low dose, such as 0.001 to 0.05, preferably0.001 to 0.01, of the systemic dose of the AGE inhibitor.

In another aspect of the invention there is provided a method for themodulation of myofibroblast activity by AGE promotion/inhibition, e.g.by administration of an AGE promoter or inhibitor. There is alsoprovided a composition for down-regulation myofibroblast activitycomprising a AGE inhibitor.

In each embodiment, repeat administrations may be necessary (e.g.injections). Furthermore, the treatment may need to be repeated tomanage or control disease progression or recurrence over a period ofmonths or years if indications of recurrence or onset are detected (oras a matter of course)

In a yet further aspect, there is provided a method and composition forthe treatment, prophylaxis or progression-inhibition of musculoskeletaladhesions by administering to a patient in need thereof a therapeutic,prophylactic or progression-inhibiting amount of a myofibroblastactivity regulator, e.g. an agent for the de-activation of myofibroblastand/or inhibiting the production of myofibroblast.

Preferably, the composition comprises, as an agent for the de-activationof myofibroblast and/or inhibiting the production of myofibroblast, oneor a combination of a TNF-α antagonist, a DAMP antagonist, an AGEinhibitor, or a DAMP and/or AGE inflammatory pathway inhibitor.Preferably, the method and composition comprises a TNF-α antagonist.Optionally, other agents may be used in such treatment including anagent of the vascular endothelial growth factor family, such as VEGF-A,VEGF-B, VEGF-C or VEGF-D or an agent encoding said VEGF or a functionalfragment thereof (such as described in WO-A-2004/082705), and/or anactivator of PPARγ (such as pioglitazone).

DAMP and/or AGE inflammatory pathway inhibitors as referred to hereininclude inhibitors or antagonists of any receptors or upstream ordownstream signalling components. For example DAMP inflammatory pathwayinhibitors may include TLR (toll like receptor) antagonists (e.g. TLR-4antagonists) or Myd88 antagonists or Myd88 down-regulators, since it isbelieved that TLR-4 and Myd88 are implicated in the DAMP mediatedinflammatory pathway. For example, AGE inflammatory pathway inhibitorsmay include RAGE inhibitors or antagonists. Optionally, according to afurther aspect of the invention, the above aspects and embodiments maybe modified by substitution or addition of TNF-α antagonist with DAMPand/or AGE inflammatory pathway inhibitors.

By musculoskeletal adhesions, it is meant a sub-set of musculoskeletalfibroproliferative disorders in which excess fibrotic tissue or scartissue is formed adjacent or in association with a tendon, muscle,joint, ligament or fascia causing an adhesion. Examples of suchmusculoskeletal adhesions include periarticular fibrosis (e.g. about theproximal interphalangeal joint) and adhesive capsulitis. Preferably,according to this aspect, there is provided a composition and treatmentfor a condition selected from perarticular fibrosis (e.g. of theproximal interphalangeal joint), spinal adhesions (e.g. post-surgical)and adhesive capsulitis.

In one particular embodiment, there is a method for the prevention ofrecurrence of Dupuytren's disease comprising administering (e.g.post-surgery, post-needle fasciotomy or after or in association withenzyme fasciotomy) a TNF-α antagonist to the nodule(s) and/or cord andadministering a TNF-α antagonist to the tissue adjacent the proximalinterphalangeal joint, whereby simultaneously treatment to preventrecurrence of Dupuytren's disease (and digital contracture) andreduction in formation and persistence of fibrotic scar tissue about thejoint can be achieved. It is believed that the effectiveness of, e.g. acollagenase treatment, of Dupuytren's disease (which suffers from a highrate of recurrence especially about the proximal interphalangeal joint)will be enhanced by co-therapy with a TNF-α antagonist (or other agentfor the de-activation of myofibroblast and/or inhibiting the productionof myofibroblast) by administering the same to clinical nodules and/orcord tissue and to subcutaneous tissue (e.g. fibrotic scar tissue)adjacent the proximal interphalangeal joint.

For adhesive capsulitis (or frozen shoulder), preferably the treatmentcomprises one or both of a TNF-α antagonist and an AGE inhibitor or DAMPantagonist.

In an alternative embodiment in each of the above mentioned aspects andembodiments, a TNF-α production or activity inhibitor may be used inplace of or together with a TNF-α antagonist.

The composition may be formulated for administration to and/or adjacentto the affected tissue (e.g. by injection, deposition during surgery orpreferably by topical application) whereby doses in the ranges describedabove in relation to musculoskeletal fibroproliferative disorders (e.g.Dupuytren's disease) are achieved/provided. Topical formulations andcombinations as described above are also included.

The invention will now be described and illustrated in more detail,without limitation, with reference to the following Examples.

EXAMPLES

The following studies were undertaken to understand better theprogression of Dupuytren's disease. Tissue was taken from nodules andcords from Dupuytren's patients and compared with non-disease palmartissue from the same patients. Studies were carried out in a cultureforce monitor developed to ensure that myofibroblast populations can bemonitored in an environment more akin to that present in diseased tissue(in line with that set out in Verjee et al, J Hand Surg Am, 34,1785-1794 and J Cell Physiol 224, 681-690). Four examples are describedbelow—Example 1 is concerned with presence, distribution and behavior ofmyofibroblast cells in diseased tissue; Example 2 is concerned with therole of inflammation in Dupuytren's disease; Example 3 examines advancedglycation end products in Dupuytren's; and Example 4 examines the roleof DAMPs in Dupuytren's.

Example 1

Over 100 Dupuytren's patient samples were collected to examinemyofibroblast distribution. Our data on >100 Dupuytren's cords show thatin the majority of patients, myofibroblasts are concentrated in nodules,located in the palm and at the level of the affected joints (see FIG.1). According to FIG. 1, nodules rich in myofibroblasts are located inthe vicinity of the finger joints. FIG. 1 shows: A: intraoperative viewof Dupuytren's cord, with location of proximal interphalangeal joint(PIPJ; 1) marked; B: Low magnification photomicrograph of histologicalsection stained for α-smooth muscle actin. A collection of α-SMA richcells in a nodule is located in the vicinity of the PIPJ; C: Highmagnification view of nodular area, showing α-SMA positive cells(myofibroblasts).

Of over 100 cords analysed, more than 60% contained nodules. Althoughthere was marked heterogeneity, nodules were very cellular, withapproximately 2.5 thousand cells per mm² arranged in whorls. On average,99% of the cells were α-SMA positive. In peri-nodular areas, there werefewer cells, approximately 800 per mm² and, on average, one third wereα-SMA positive.

FIG. 2 shows that nodules are mostly cellular and are rich in α-SMApositive cells. Examination of 24 Dupuytren's patient samples byelectron microscopy showed that clinical recurrence was not related topatient age at onset, duration, or severity of disease. Histologicalnodules were seen as frequently in samples from both primary andrecurrent disease (two-thirds of cords in each case) and there was alsono significant difference in digital contracture between primary andrecurrent disease. Furthermore, there was also no difference in nodularsurface area between primary dermofasciectomy samples, primaryfasciectomy, secondary fasciectomy or dermofaciectomy followingrecurrent disease (p=0.5). A similar pathogenesis in both primary andrecurrent disease is likely and nodularity is unlikely to bedown-regulated following previous surgery. Indeed, the increased motionfollowing initial surgery may facilitate myofibroblast differentiationand persistence. It is possible that residual unexcised Dupuytren'stissue following fasciotomy or fasciectomy and firebreakdermofasciectomy, may serve as a trigger for recurrence. The persistenceof myofibroblasts may explain the high recurrence rates seen followingsurgical fasciotomy or collagenase injection. Therefore, a key elementof preventing recurrent disease may be to down regulate the remainingmyofibroblasts.

95% (36/38) of nodules were in the vicinity of the PIPJ and nodules werealso observed over the MCPJ in the only two cases markedintra-operatively for the MCPJ and one case marked for the DIPJ. Inearly or active disease, tension may act intermittently on Dupuytren'stissue as active extension of the PIPJ offers resistance against thethickened, contracted palmar fascia. The increased tension sensed bycells may promote myofibroblast differentiation through recruitment ofα-SMA to stress fibres and specialised attachment site formation understrict control of TGF-β1. This in turn leads to greater forcegeneration. A densely packed cellular nodule could then theoreticallyexert sufficient force to promote or sustain digital contracture. Thecells then remodel the surrounding matrix to a more shortenedconfiguration. The resulting increased flexion deformity would impairfunction and the reduced movement at the joint would in turn lead to areduction in tension sensed by nodular myofibroblasts. It is possiblethat with advanced digital contractures, reduced tension through limitedactive joint extension may lead to myofibroblast apoptosis, wherebymyofibroblast rich nodules fail to persist. This may explain theprogression from nodular to non-nodular cords and would also explain whypatients with non-nodular cords tended to have more severe flexiondeformities. Thus, myofibroblast aggregation in nodules in the vicinityof joints may lead to digital contracture and with subsequent matrixremodelling result in shortening of the affected fascia. Eventuallyfixed flexion deformity develops leading to an altered mechanicalenvironment with loss of tension, myofibroblast apoptosis and thus mayexplain residual non-nodular cords. It can be concluded that themyofibroblast phenotye depends on tension in the surrounding matrix.

The culture force monitor (CFM) utilised and culture conditions areshown in FIG. 3: (A) Rectangular seeded collagen gels were cast andfloated in medium, tethered between two flotation bars one of which isheld stationary whilst the other is attached to a force transducer. (B)Cell-generated tensional forces in the collagen gel are detected by theforce transducer, and live data are logged every 15 seconds providing acontinuous output of force (dynes, 1×10⁵ N) generated. (C) After 24 hourcontraction, gels are harvested and processed for α-SMA mRNA, proteinand immunofluorescence. (D) Cells were routinely seeded in gels with ahigh aspect-ratio collagen lattice, although low aspect-ratio lattices(E) were also used in experiments to compare effects of less strain oncell contractility.

Surgically excised cords were bisected and half processed for cellculture, whilst the cut surface of the mirror half was processed toidentify samples with α-SMA-rich nodules (condensation of cells) byimmunohistochemistry. Subsequent quantification by immunofluorescencedemonstrated on average 35% of cells expressed α-SMA stress fibres inhistology confirmed nodular samples, as compared to 10% α-SMA stressfibres in non-nodular samples. Although this still does not constitute ahomogenous population of myofibroblasts, this method of samplingα-SMA-rich cells represents a significant improvement on previousstudies, which have reported on average between 9.7% and 15%α-SMA-positive cells isolated from clinical and not histologicallydefined nodules. 1-4% of dermal fibroblasts were found to have α-SMApositive stress fibres.

FIG. 4 shows isometric contraction of collagen gels by dermalfibroblasts and Dupuytren's nodule-derived cells. Collagen gels wereseeded with 1.5 million non-palmar fibroblasts (A), palmar fibroblasts(B) or Dupuytrens nodule-derived cells (C), cultured for 24 h in the CFMand real-time isometric force contraction was quantified. Data shownrepresent triplicate experiments using cells derived from one patient.Dermal fibroblasts in fibroblast populated collagen lattices (FPCL) inour CFM reached a plateau, whereas nodule-derived cells continued tocontract in a dose-dependent manner over a 24 hour test period.

FIG. 5 shows that cells isolated from nodules had a much higher rate ofcontraction measured as the average rate of contraction between 6 and 24hours in the CFM compared to palmar or non-palmar dermal fibroblasts.High contractility is one of the characteristics of myofibroblasts andis responsible for digital contracture in Dupuytren's disease.

In FIG. 6, it is shown that the contractility of Dupuytren'snodule-derived cells is regulated by post-transcriptional changes inα-SMA: (A) [rate of contraction (dynes/hr)] Isometric force in collagengels with nodule-derived myofibroblasts (nodule), non-palmar (NPS) andpalmar dermal fibroblasts (PS) over 24 hours (±SEM). After 24 h (B)α-SMA mRNA was compared to RPLPO by quantitative RT-per, (C) α-SMA mRNAcompared to GAPDH and (D) α-SMA protein compared with vimentin.Experiments were performed in triplicate and data are shown as the mean(±SEM) from a total of 3 different nodular and non-nodular matchedpatient samples

After harvesting FPCLs following 24 hours contraction in the CFM,comparisons were made between α-SMA mRNA levels, α-SMA proteinexpression and α-SMA protein localisation by immunofluorescence in thematched cell types. No differences in α-SMA mRNA levels were seenbetween nodule-derived cells and dermal fibroblasts, althoughapproximately 3-fold greater α-SMA protein levels were seen innodule-derived cells compared with matched dermal fibroblasts.Furthermore, using immunofluorescence we found that in dermalfibroblasts, α-SMA was typically distributed in a ‘halo’ within theperi-nucleur cytoplasm, whereas in nodule-derived cells, α-SMA wasfrequently localised in stress fibres throughout the cell processes upto cell-matrix attachment sites, as can be seen in FIG. 6 E.

Cells were also cultured on glass coverslips for 24 hours, fixed andthen immunofluorescently labelled using α-SMA antibodies (red),phalloidin (green) and DAPI (blue). Our immunofluoresence datademonstrate that palmar and non-palmar fibroblasts when cultured inmonolayer acquired a proto-myofibroblast phenotype, with the expressionof de novo cytosolic α-SMA. In contrast, significantly moredifferentiated myofibroblasts with α-SMA incorporated to stress fibreswere seen in nodule-derived cells. These differences seen betweennodule-derived, non-palmar and palmar skin cells from matched sampleshave not been previously reported. We simultaneously examined α-SMAprotein levels, protein localization and mRNA levels in cells isolatedfrom the same patient. Our findings suggest that post-transcriptionalchanges in α-SMA occur in genetically matched cells to mediate theDupuytren's myofibroblast cell phenotype.

Example 2 Role of Inflammation in Dupuytren's Disease

The nodules were then examined for the presence of other cell types,specifically inflammatory cells. We found that large numbers of bothmacrophages and mast cells were present in nodules but not innon-nodular regions of the cords.

FIG. 7 shows inflammatory cells in Dupuytren's nodule and cord. Digitalcord sections were serially stained for α-SMA, CD68 positive macrophagesand mast cell tryptase. The images are representative of 15 patientsamples.

We systematically quantified the number of inflammatory cells observedthroughout excised Dupuytren's cord tissue in 10 patient samples. Foreach region (nodule, cord distal to nodule and non-nodular cord), thetotal number of cells, the number of α-SMA positive cells and cellsstained for neutrophil elastase, mast cell tryptase, CD3positive Tcells, CD 4 positive T cells, CD68 positive macrophages were counted(×20 magnification) (Table 1).

TABLE 1 Quantification of total cell number: α-SMA positive cells, CD3positive T cells, CD4 positive T cells, CD68 positive macrophages, andcells positive for mast cell tryptase and neutrophil elastase throughoutexcised Dupuytren's cord. Nodules, cord distal to nodule and non-nodularcord were analysed (presented as mean count (±SDEV) per mm²) Six fieldsof view were counted within each region. Non-nodular Nodule Distal tonodule cord IHC stain mean SDEV mean SDEV mean SDEV Total cells 1515 181416 104 504 163 α-SMA 1493 199 12 8 8 7 Neutrophil elastase 2 1 0 1 0 0CDS positive T cells 220 99 2 2 1 2 CD4 positive T cells 2 1 0 0 0 0CD68 positive 282 54 1 1 1 1 macrophages Mast cell tryptase 48 11 1 1 01

These data show that CD68 positive macrophages, CD3 T-cells and mastcell tryptase positive cells were common within cellular nodules andsparse within cord tissue. Neutrophil elastase positive cells and CD4positive T-cells were observed infrequently throughout Dupuytren'stissue. Dupuytren's nodules contained numerous mast cells, which are arich source of TNF-α (Krishnaswamy et al., 2006). Whilst Dupuytren'snodular tissue is populated with highly contractile myofibroblasts, thepresence of inflammatory cells suggests that inflammation may beimportant in pathogenesis of the disease. In non-nodular cord, almost noinflammatory cells were observed and it is also of interest thatvirtually no cells stained positive for neutrophil elastase in eithernodular or non-nodular cord. This is in contrast to inflammation duringwound healing, where neutrophils are commonly seen and they are involvedwith clearance of debris and bacteria and initiatingmyofibroblast-dependent wound contraction. However, it is important tonote that whilst excised digital cord samples contain nodules, they donot necessarily reflect the processes at the earliest stages of thedisease.

Example 3 Advanced Glycation End Products and their Receptor

We examined the distribution of RAGE in Dupuytren's tissue and bothpalmar and non-palmar skin. We found abundant staining for RAGE inDupuytren's nodules, where it co-localised with the myofibroblasts (seeFIG. 8). Digital cord samples were longitudinally bisected and fixed informalin. Histological sections were taken from the cut surface of cordand serial sections were stained for (A, C) α-SMA and (B, D) RAGEantibodies. Scale bars as shown. Images are representative from 15patient samples. RAGE co-localises with α-SMA distribution inDupuytren's nodules.

We also found increased staining for RAGE in the superficial layers ofthe epidermis in palmar skin compared to non-palmar skin and FACSstaining showed significantly higher RAGE expression by dermalfibroblasts from palmar skin compared to non-palmar skin. See FIG. 9.

Non-palmar and palmar skin samples were fixed in formalin. Histologicalsections were stained for RAGE. (A, C) Non-palmar skin and (B, D) palmarskin. Scale bars are shown. Images are representative from 6 matchedpatient samples. FIG. 9 illustrates the differential distribution ofRAGE within non-palmar and palmar skin.

We also demonstrated that nodule-derived cells express higher levels ofcell surface RAGE than matched dermal fibroblasts. Nodule-derived cells,palmar and non-palmar fibroblasts (1×10⁴ cells per experiment) werestained with RAGE antibody, fluorescently labelled and mean fluorescenceintensity assessed by FACS analysis. (FIG. 10 A,C) Cell surface RAGEexpression levels in nodule-derived cells as compared with matcheddermal fibroblasts. (B,D) Cell surface RAGE expression levels innon-nodular cells as compared with matched dermal fibroblasts. Resultsin A and B are shown for 4 matched nodular patient and non-nodularsamples (±SEM). * represents p=0.01. (E) RAGE fluorescent intensitytrace showing nodule-derived cells, non-palmar fibroblasts, palmarfibroblasts and isotype control from 1 representative nodular matchedpatient sample, and (F) from 1 representative non-nodular matchedpatient sample. See FIG. 10.

We demonstrated that RAGE cell surface expression is greater in palmarthan non-palmar fibroblasts. Fibroblasts (1×10⁴) from matched palmar andnon-palmar skin were stained with RAGE antibody, fluorescently labelledand the mean fluorescence intensity analysed by FACS. Cell surface RAGEexpression levels were consistently higher in palmar fibroblasts thannon-palmar fibroblasts. Data are shown from 8 matched patient samples.See FIG. 11.

In a further experiment to investigate the effect of AGE onmyofibroblast formation, collagen gels were seeded with 1.5 millionpalmar fibroblasts and cultured for 24 h in the absence (PS alone) orpresence of bovine serum albumin (BSA) (15 μg/ml), or AGEs-BSA (150μg/ml) for varying periods and isometric force contraction quantified inthe culture force monitor. Data are shown as +/−SEM from triplicateexperiments with samples from 3 different patients in FIG. 14. It isapparent from FIG. 14 that contractility of palmar dermal fibroblasts isnot affected by exposure to AGEs.

We went on to investigate whether advanced glycation end products mayalso act via inflammatory cells and in other systems have been shown tolead to pro-inflammatory cytokine release (Uribarri et al., 2005).Collagen gels seeded with palmar fibroblasts were cultured for 24 h withsupernatants from AGE-(100μ/ml) stimulated monocytes (M) in the absenceor presence of anti TNF-α (10 μg/ml) and isometric force contractionquantified. Experiments were performed in duplicate. Interestingly thesupernatant from human monocytes co-cultured with AGEs stimulated palmarfibroblast contraction in a TNF-α dependent manner (FIG. 20).

Example 4

We examined the effect of addition of exogenous HMGB1 to palmarfibroblasts. Collagen gels seeded with palmar fibroblasts were culturedfor 24 h in the absence (PS alone) or presence of TNF-α (1 ng/ml), orHMGB1 (1 ng/ml) or TGF-β1 (10 ng/ml) and isometric force contractionquantified (utilising the culture force monitor technique, such asdescribed in Verjee et al, Hand Surg Am, 34, 1785-1794, 2009 and Verjeeet al, J Cell Physiol, 2010). Data from the experiment are shown in FIG.12 as +/−SD from triplicate experiments (except HMGB1 which isduplicate).

Whilst there was a trend towards increased contraction resulting fromHMGB1, this was not statistically significant (FIG. 12). Our data showedsignificant increased palmar fibroblast contractility (p=0.0001) with 10ng/ml TGF-β1 compared to untreated palmar fibroblasts.

We found that human monocytes exposed to S100A8 and to some extentS100A9 (other Alarmins) produced TNF-α in a dose dependent manner, as isillustrated by FIG. 15 As can be seen from FIG. 15, S100A8 is moreactive than S100A9 and S100A12 within the tested range. LPS, a pathogenassociated molecular pattern (PAMP) is shown as positive control.

The known receptors for S100A8 are the receptor for advanced glycationend products (RAGE) and the Toll-like receptors 2 and 4. Human monocytesat 1×10⁵/ml were incubated in 10% FCS with human 5100 A8 at 0.5 μg/mlwith the addition of either antibody to TLR4, TLR2 or isotype controls(not shown), or soluble RAGE (sRAGE)) over 14 hours. TNF-α levels weredetermined by ELISA. We have found that the predominant receptor forbinding S100A8 on monocytes leading to TNF-α production is TLR-4 and notRAGE or TLR-2 (FIG. 16).

We have confirmed that S100A8 predominantly binds to TLR-4 and that theintracellular signalling leading to TNF-α production by monocytes isentirely dependent on adaptor protein MyD88 by comparing the effect ofmurine S100A8 on TNF-α production by bone marrow cells derived fromTLR-4 or MyD88 deficient mice with bone marrow derived cells fromwild-type C57Bl/6 animals (FIG. 17). In FIG. 17, TNF-α produced bymurine bone marrow cells of wild type, TLR4 and MyD88 mice on exposureto murine S100A8 were measured by ELISA.

It is more difficult to show in vitro that HMGB1 also acts on monocytesto lead to pro-inflammatory cytokine release. This is because in vivo itacts in conjunction with other TLR ligands and highly purified HMGB1alone does not lead to TNF-α production by monocytes in vitro (FIG. 18).Our experiment, the results of which are shown in FIG. 18, involvedhuman monocytes at 1×10⁵/ml incubated in 10% FCS with HMGB1 or LPS aloneor together at concentrations shown over 14 hours. TNF-α levels weredetermined by ELISA. It was found that HMGB1 alone does not stimulateTNF-α production by monocytes but is active in combination with LPS.

FIG. 19 shows a schematic of proposed mechanism of the role of traumaand alarmins in the pathogenesis of Dupuytren's disease. As can be seen,trauma (101) causes cell injury (103) and consequent release of Alarmins(105) such as S100A8, which binds to TLR-4 (107) in an inflammatory cellsuch as a macrophage (109) causing pro-inflammatory cytokines such asTNF-α to be produced (113) signalled via Myd88 (111). TNF-α may thenbind to TNFR (115) on fibroblast precursor (117) resulting in formationof myofibroblast (119).

Example 5

Primary passage cultured cells from a Dupuytren's nodule (comprisingmyofibroblast cells) were treated, using the culture force monitordescribed above, with a monoclonal human TNF-α antibody (monoclonal IgG₁culture #1825, as available from R&D Systems of Canada) in an amount of10 μg/ml. Compared with a control culture of such primary passage cells,the anti-TNF-α treated cells were found over 24 hours to contract by anamount of greater than 30% less than control (which it is believedcorresponds to effective myofibroblast deactivation of greater than 30%compared with control). This is shown in FIG. 13, where the gradient (orrate of contraction; in Dynes/h) over 24 hours is illustrated for eachof the control cells and the TNF-α antibody treated cells.

This directly shows that myofibroblast cells cultured from a clinicalfrom a Dupuytren's disease patient has reduced activity (e.g. reducedcontractile behaviour and/or reduced abundance) when treated with aTNF-α antagonist, even over only 24 hours. Since the effect of the TNF-αantagonist on myofibroblasts in the clinical situation will be ongoingand the therapeutic regime in a patient may involve repeat applications,it is believed that this experiment shows that myofibroblast activitycan be effectively managed, thereby reducing the progression of and/orinhibiting the recurrence of musculoskeletal fibroproliferativedisorders and, in particular, Dupuytren's disease by local applicationof a TNF-α antagonist to the disease site.

We were able to confirm that addition of TGF-β1 human palmar fibroblastsfrom in a collagen lattice under isometric conditions enhancedcontractility (see FIG. 21). Collagen gels seeded with palmarfibroblasts were cultured for 24 h in the absence (PS alone) or presenceof TGF-β1 (10 ng/ml). Data are shown as +/−SD from triplicateexperiments using cells from 3 patients.

We then compared the effect of TNF-α on the rate of contraction onpalmar skin and non-palmar skin from a Dupuytren's patient. Collagengels seeded with palmar fibroblasts were cultured for 24 h in theabsence (PS or NPS alone) or presence of TNF-α (1 ng/ml). Data are shownas +/−SD from triplicate experiments using cells from 3 patients forpalmar skin and one patient non-palmar skin (** p=0.0012, ns=notsignificant). We found significantly enhanced contraction in the cultureforce monitor (FIG. 22) on addition of TNF-α to the palmar skinfibroblasts. However, there was no change, or a slight reduction, incontraction rate when TNF-α was added to non-palmar dermal fibroblastsalso obtained from patients undergoing dermofasciectomy for Dupuytren'sdisease. It is interesting to note that Dupuytren's disease only affectsthe palms of the hand and rarely the soles of the feet or the tunicaalbuginea of the penis (Peyronie's disease).

The key next question was whether the contractility of myofibroblasts inDupuytren's disease could be reversed by the addition of anti-TNF-α in adose-dependent manner. Collagen gels seeded with 1.5 million Dupuytren'smyofibroblasts/fibroblasts were cultured for 24 h in the presence ofanti TNF-α (murine anti-human R&D Systems, MAB2010) and isometric forcecontraction quantified. Experiments were performed in triplicate usingcells from 5 consecutive unselected patients. There was no effect withisotype control antibody or with 0.1 μg/ml of anti TNF-α. Valuesrepresent mean±SEM. The results are shown in FIG. 23. Addition ofanti-TNF-α at a dose range of 1-10 μg/ml to myofibroblasts fromDupuytren's cord down-regulated their contraction in the culture forcemonitor in a dose-dependent manner (FIG. 23).

We next assessed the effect of anti-TNF-α on myofibroblast morphology.All the cells in the untreated gels or those exposed to IgG isotypecontrol antibody were spindle shaped and aligned in the axis of maximalstress (FIG. 24 a). However, in the gels treated with 10 μg/mlanti-TNF-α, many of the cells showed a stellate morphology, without anyalignment to the direction of stress (FIG. 24 b,c).

For the experiments for FIG. 24, gels from the experiments shown in FIG.23 were fixed in 3% paraformaldehyde were immunofluorescently labelledusing α-smooth muscle actin antibodies (red), phalloidin (green) andDAPI (nuclei-blue). FIG. 24 a shows cells from a gel exposed to isotypecontrol IgG antibody. FIGS. 24 b and c show cells from a gel exposed to10 μg/ml anti-TNF-α antibody stained with phalloidin and antibody toα-smooth muscle actin respectively. Original images photographed at×100.

FIG. 25 illustrates a schematic of proposed role of advanced glycationend products, injury and alarmins in the pathogenesis offibroproliferative disorders, highlighting the key role of TNF-α in thefinal common pathway. Hence TNF-α is a key therapeutic target for bothearly Dupuytren's disease and to prevent recurrence following treatmentwith collagenase.

These Examples illustrate initial findings that enhance understanding ofDuputren's nodule material and contractile behaviour especially relatingto the role and behaviour of active myofibroblasts. These findingsimplicate TNF-α in myofibroblast activity as well as DAMPs and AGE,which support the finding that TNF-α antagonists, DAMP antagonistsand/or AGE inhibitors may be used to prevent or inhibit disease onset orprogression from early state disease to established state disease and toprevent or inhibit disease recurrence in established disease wherepatients have undergone a primary corrective treatment.

The invention has been described with reference to a preferredembodiment. However, it will be appreciated that variations andmodifications can be effected by a person of ordinary skill in the artwithout departing from the scope of the invention.

The invention claimed is:
 1. A method for treating a patient with earlydisease state Dupuytren's disease comprising injecting an amount of aTNF-α antagonist effective to treat the patient directly into one ormore clinical or histological nodules of the patient.
 2. A methodaccording to claim 1, wherein the TNF-α antagonist is selected from oneor more of Infliximab, Adalimumab, Certolizumab pegol, Golimumab orEtanercept.
 3. A method according to claim 1, wherein the TNF-αantagonist is an anti-TNF-α antibody.
 4. A method according to claim 1,wherein the TNF-α antagonist is injected into clinical nodules.
 5. Amethod according to claim 1, wherein the TNF-α antagonist is Infliximab.6. A method according to claim 1, wherein the TNF-α antagonist isAdalimumab.
 7. A method according to claim 1, wherein the TNF-αantagonist is Certolizumab pegol.
 8. A method according to claim 1,wherein the TNF-α antagonist is Golimumab.
 9. A method according toclaim 1, wherein the TNF-α antagonist is Etanercept.